邻近标记技术对活体小鼠耳蜗毛细胞表面蛋白进行特异性标记

In vivo proximity labeling of cell-surface proteins in mouse cochlear hair cells

摘要

目的 开发标记小鼠耳蜗毛细胞的细胞表面蛋白的方法 , 为测定耳蜗毛细胞表面蛋白质组提供依据 。方法 构建在毛细胞中特异性表达一种辣根过氧化物酶融合蛋白 ( HRP fused with a transmembrane domain , HRP-TM ) 的重组腺相关病毒 ( recombinant adeno-associated virus , rAAV ), HRP-TM 可以定位到毛细胞膜的外表面 。通过显微注射将病毒注射到 2 d 新生 C57BL/6 小鼠的后半规管中 , 30 d 时检测听觉脑干反应 ( auditory brainstem response , ABR ), 并通过基底膜免疫荧光染色检测耳蜗中 HRP 表达和蛋白标记情况 。结果 ABR 结果显示 , 注射病毒的小鼠和未注射病毒的对照小鼠相比 , 听力无明显差异 。基底膜免疫荧光染色结果显示 , HRP-TM 在毛细胞中特异性表达 , 内毛细胞的病毒感染效率超过 95% , 外毛细胞的病毒感染效率达到 80% 左右 。而且 , 毛细胞的形态和排列与对照相比无明显差异 。用荧光偶联链霉亲和素进行免疫荧光染色 , 只有表达 HRP-TM 的毛细胞的细胞膜表面有明显荧光信号 。结论 本研究中构建的 rAAV 病毒可以高效特异性的标记耳蜗毛细胞的细胞表面蛋白 , 并且无明显的细胞毒性 , 不影响毛细胞的正常生理功能 , 为下一步分离耳蜗毛细胞的细胞表面蛋白并进行质谱分析奠定了基础 。

Abstract

Objective To develop a method for labeling cell-surface proteins of cochlear hair cells in mice and establish conditions for profiling the cell-surface proteome of these cells. Methods A recombinant adeno-associated virus (rAAV) was engineered to express a horseradish peroxidase fusion protein (HRP-TM) specifically in hair cells, targeting the outer surface of the cell membrane. The virus was microinjected into the posterior semicircular canal of 2-day-old C57BL/6 mice. At 1 month of age, auditory brainstem response (ABR) was measured, and HRP expression and protein labeling were assessed via basilar membrane immunofluorescence staining. Results ABR results showed no significant hearing differences between virus-injected and control mice. Immunofluorescence revealed HRP-TM was specifically expressed in hair cells, with >95% transduction efficiency in inner hair cells and ~80% transduction efficiency in outer hair cells. Hair cell morphology and arrangement remained normal. Fluorescent streptavidin staining confirmed membrane-specific labeling exclusively in HRP-TM-expressing hair cells. Conclusion The rAAV-HRP-TM system efficiently and specifically labels cochlear hair cell surface proteins without cytotoxicity or functional impairment, providing a foundation for isolating and analyzing these proteins via mass spectrometry.

关键词

耳蜗毛细胞 / 邻近标记 / 重组腺相关病毒 / 辣根过氧化物酶 / 细胞表面蛋白质组

Key words

Cochlear hair cells / Proximity labeling / rAAV / HRP / Cell-surface proteome

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基金资助

山东省自然科学基金(ZR2023MC207)